This invention relates to a novel method of screening for inhibitors of beta-amyloid production, and thereby identifying such inhibitors as therapeutics for neurological and other disorders involving APP processing and beta-amyloid production. This invention also relates to identifying macromolecules involved in APP processing and beta-amyloid production. Furthermore, inhibitors identified by the screening method of the present invention are useful in the treatment of neurological disorders, such as Alzheimer""s disease, which involve elevated levels of Axcex2 peptides.
Alzheimer""s disease (AD) is a degenerative brain disorder characterized clinically by progressive loss of memory, temporal and local orientation, cognition, reasoning, judgment and emotional stability. AD is a common cause of progressive dementia in humans and is one of the major causes of death in the United States. AD has been observed in all races and ethnic groups worldwide, and is a major present and future health problem. No treatment that effectively prevents AD or reverses the clinical symptoms and underlying pathophysiology is currently available (for review, Dennis J. Selkoe; Cell Biology of the amyloid (beta)-protein precursor and the mechanism of Alzheimer""s disease, Annu Rev Cell Biol, 1994, 10: 373-403).
Histopathological examination of brain tissue derived upon autopsy or from neurosurgical specimens in effected individuals revealed the occurrence of amyloid plaques and neurofibrillar tangles in the cerebral cortex of such patients. Similar alterations were observed in patients with Trisomy 21 (Down""s syndrome), and hereditary cerebral hemorrhage with amyloidosis of the Dutch-type.
Neurofibrillar tangles are nonmembrane-bound bundles of abnormal proteinaceous filaments and biochemical and immunochemical studies led to the conclusion that their principle protein subunit is an altered phosphorylated form of the tau protein (reviewed in Selkoe, 1994).
Biochemical and immunological studies revealed that the dominant proteinaceous component of the amyloid plaque is an approximately 4.2 kilodalton (kD) protein of about 39 to 43 amino acids. This protein was designated Axcex2, xcex2-amyloid peptide, and sometimes xcex2/A4; referred to herein as Axcex2. In addition to deposition of Axcex2 in amyloid plaques, Axcex2 is also found in the walls of meningeal and parenchymal arterioles, small arteries, capillaries, and sometimes, venules. Axcex2 was first purified, and a partial amino acid reported, in 1984 (Glenner and Wong, Biochem. Biophys. Res. Commun. 120: 885-890). The isolation and sequence data for the first 28 amino acids are described in U.S. Pat. No 4,666,829.
Compelling evidence accumulated during the last decade revealed that Axcex2 is an internal polypeptide derived from a type 1 integral membrane protein, termed xcex2 amyloid precursor protein (APP). xcex2 APP is normally produced by many cells both in vivo and in cultured cells, derived from various animals and humans. Axcex2 is derived from cleavage of xcex2 APP by as yet unknown enzyme (protease) system(s), collectively termed secretases.
The existence of at least four proteolytic activities has been postulated. They include xcex2 secretase(s), generating the N-terminus of Axcex2, xcex1 secretase(s) cleaving around the 16/17 peptide bond in Axcex2, and xcex3 secretases, generating C-terminal Axcex2 fragments ending at position 38, 39, 40, 42, and 43 or generating C-terminal extended precursors which are subsequently truncated to the above polypeptides.
The gene encoding a human aspartic protease that cleaves the xcex2-secretase site of xcex2-amyloid precursor protein has recently been isolated; this gene and encoded protein is designated as BACE (Vassar et al., Science (1999) 286: 735-741) or as memapsin-2 (Lin et al., PNAS (2000) 97: 1456-1460) and is designated herein as xe2x80x9cBACE/memapsin-2xe2x80x9d.
Several lines of evidence suggest that abnormal accumulation of Axcex2 plays a key role in the pathogenesis of AD. Firstly, Axcex2 is the major protein found in amyloid plaques. Secondly, Axcex2 is neurotoxic and may be causally related to neuronal death observed in AD patients. Thirdly, missense DNA mutations at position 717 in the 770 isoform of xcex2 APP can be found in effected members but not unaffected members of several families with a genetically determined (familiar) form of AD. In addition, several other xcex2 APP mutations have been described in familial forms of AD. Fourthly, similar neuropathological changes have been observed in transgenic animals overexpressing mutant forms of human xcex2 APP. Fifthly, individuals with Down""s syndrome have an increased gene dosage of xcex2 APP and develop early-onset AD. Taken together, these observations strongly suggest that Axcex2 depositions may be causally related to the AD.
It is hypothesized that inhibiting the production of Axcex2 will prevent and reduce neurological degeneration, by controlling the formation of amyloid plaques, reducing neurotoxicity and, generally, mediating the pathology associated with Axcex2 production. One method of treatment methods would therefore be based on drugs that inhibit the formation of Axcex2 in vivo.
Methods of treatment could target the formation of Axcex2 through the enzymes involved in the proteolytic processing of xcex2 amyloid precursor protein. Compounds that inhibit xcex2 or xcex3 secretase activity, either directly or indirectly, could control the production of Axcex2. Advantageously, compounds that specifically target xcex3 secretases, could control the production of Axcex2. Such inhibition of xcex2 or xcex3 secretases could thereby reduce production of Axcex2, which, thereby, could reduce or prevent the neurological disorders associated with Axcex2 protein.
It is believed that several macromolecules, some of which have proteolytic activity, are involved in the processing of amyloid precursor protein (APP). This processing leads to several products including the xcex2-amyloid peptides (Axcex2) believed etiologically important in Alzheimers Disease. We have discovered novel tagged compounds, functional in themselves as Axcex2 inhibitors, for use in identifying a site or sites on one or more macromolecules critical to the processing of xcex2 APP and the production of Axcex2. We have discovered novel tagged compounds which inhibit the proteolytic activity leading to production of Axcex2 by interacting with one or more macromolecules critical to the processing of APP and the production of Axcex2. We have also discovered a site of action of these tagged compounds using radioisotope tagged derivatives of a compound of Formula (I).
Three examples of tagged compounds include (I-7T), (I-11T), and (I-43T): 
(I-7) R**=1H; Y=xe2x80x94Oxe2x80x94;
(I-7T): R**=3H; Y=xe2x80x94Oxe2x80x94; and
(I-11): R**=1H; y=xe2x80x94C(xe2x95x90O)xe2x80x94;
(I-11T): R**=3H; Y=xe2x80x94C(xe2x95x90O)xe2x80x94; and 
(I-43): R**=1H;
(I-43T): R**=3H.
The concentration of Compound (I-7) leading to half-maximal inhibition (IC50) of proteolytic activity leading to Axcex2 production in HEK293 cells expressing APP 695 wt is similar to the concentration leading to half-maximal inhibition (IC50) of Compound (I-7T) binding to membranes derived from the same cell line. The correlation holds for compounds (I-11T) and (I-43T). Also using a compound of Formula (I), we have discovered a macromolecule containing a binding site of action for compounds of Formula (I) critical to the processing of APP and the production of Axcex2.
Furthermore, we have discovered through competitive binding studies that there is a good correlation between the ability of a series of compounds to inhibit the proteolytic activity leading to production of Axcex2 and to inhibit the binding of Compound (I-7T), (I-11T), or (I-43T) to said membranes. Thus, the binding of Compound (I-7T), (I-11T), or (I-43T) to relevant tissues and cell lines, membranes derived from relevant tissues and cell lines, as well as isolated macromolecules and complexes of isolated macromolecules, is useful in the identification of inhibitors of Axcex2 production through competitive binding assays. Furthermore, such competitive binding assays are useful in identification of inhibitors of proteolytic activity leading to Axcex2 production for the treatment of Alzheimer""s disease. Furthermore, such competitive binding assays are useful in identification of inhibitors of proteolytic activity leading to Axcex2 production for the treatment of neurological disorders and other disorders involving Axcex2, APP, and/or Axcex2/APP associated macromolecules, and other macromolecules associated with the site of Compound (I-7T), (I-11T), or (I-43T) binding.
One object of the present invention is to provide a novel method of screening for inhibitors of beta-amyloid production, and thereby identifying such inhibitors as therapeutics for neurological and other disorders involved in APP processing and beta-amyloid production. The method comprises 1) contacting a potential inhibitor of beta-amyloid production and a tagged inhibitor of beta-amyloid production with at least one macromolecule involved in the processing of APP and the production of beta-amyloid peptide, said macromolecule containing a binding site specific for said tagged inhibitor of beta-amyloid production; 2) separating the tagged inhibitor of beta-amyloid production bound to said macromolecule from the tagged inhibitor of beta-amyloid production free from said macromolecule; and 3) determining an inhibitory concentration of the potential inhibitor of beta-amyloid production from the concentration of tagged inhibitor of beta-amyloid production bound to said macromolecule.
It is another object of the present invention to provide the use of a tagged inhibitor of beta-amyloid production to identify macromolecules involved in APP processing.
It is another object of the present invention to provide the macromolecules involved in APP processing which a tagged inhibitor of beta-amyloid production binds to specifically.
It is another object of the present invention to provide the use of macromolecules involved in APP processing, which a tagged inhibitor of beta-amyloid production binds to specifically, for the identification of inhibitors as therapeutics for neurological and other disorders involved in APP processing and beta-amyloid production.
It is another object of the present invention to provide the use of macromolecules involved in APP processing, which a tagged inhibitor of beta-amyloid production binds to specifically, for the assaying of inhibitors of beta-amyloid production.
It is another object of the present invention to provide pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an inhibitor of beta-amyloid production, or a pharmaceutically acceptable salt or prodrug form thereof, identified by the screening assay of the present invention.
It is another object of the present invention to provide a method for treating degenerative neurological disorders involving beta-amyloid production, including Alzheimer""s disease, comprising administering to a host in need of such treatment a therapeutically effective amount of an inhibitor of beta-amyloid production, or a pharmaceutically acceptable salt or prodrug form thereof, identified by the screening assay of the present invention.
It is another object of the present invention to provide an inhibitor of beta-amyloid production which interacts with the binding site for a compound of Formula (I-7T) on a macromolecule involved in the production of beta-amyloid peptide.
These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors"" discovery that compounds of Formula (I): 
(I-7): R**=1H; Y=xe2x80x94Oxe2x80x94;
(I-7T): R**=3H; Y=xe2x80x94Oxe2x80x94; and
(I-11): R**=1H; Y=xe2x80x94C(xe2x95x90O)xe2x80x94;
(I-11T): R**=3H; Y=xe2x80x94C(xe2x95x90O)xe2x80x94; and 
(I-43): R**=1H;
(I-43T): R**=3H;
bind specifically to a binding site on a macromolecule or a complex of macromolecules involved in APP processing to produce reduction of Axcex2 peptide production. For example, the concentration of Compound (I-7) leading to half-maximal inhibition (IC50) of Axcex2 production in HEK293 cells expressing APP 695 wt is similar to the concentration leading to half-maximal inhibition (IC50) of Compound (I-7T) binding to membranes derived from the same cell line.
It is another object of the present invention to provide radiolabeled inhibitors of APP processing and/or the production of beta-amyloid production for use in methods of in vivo diagnostic imaging in the diagnosis of diseases involving APP processing and/or the production of beta-amyloid production. Also provided in the present invention are methods of in vivo diagnostic imaging comprising administering to a subject a diagnostically effective amount of a radiolabeled inhibitor of APP processing and/or the production of beta-amyloid production